Targeted gene silencing using RGD-labeled chitosan nanoparticles

Abstract

Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA).

Experimental design: RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with alphanubeta3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma.

Results: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the alphanubeta3 integrin-positive tumor endothelial cells in the A2780 tumor-bearing mice. This approach resulted in significant inhibition of tumor growth compared with controls.

Conclusions: This study shows that RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential for broad applications in human disease.

Fig. 1

A, Conjugation of RGD to chitosan (CH). Physical properties of siRNA/RGD-CH-NPs. B (upper panel), RGD concentration in the siRNA/RGD-CH-NPs was calculated by measuring FITC intensity based on a calibration curve of standard concentration of FITC-labeled with RGD by fluorescence spectrophotometry. B (middle and lower panel), Size and zeta potential of siRNA/RGD-CH-NPs were measured by light scattering with a particle size analyzer and Zeta Plus, respectively. C, Incorporation of FITC-labeled RGD (green) and Alexa555 siRNA (red) into siRNA/RGD-CH-NPs was observed by fluorescence microscopy (magnification ×400, upper panel, scale bar: 1 μm). Morphology of siRNA/RGD-CH-NP 5 was examined by scanning electron microscopy (SEM, lower panel). Error bars represent s.e.m. *p<0.05.

Fig. 2

Binding of siRNA/RGD-CH-NPs against ovarian cancer cells in vitro. A, ανβ3 integrin expression on SKOV3ip1 or A2780ip2 cells by flow cytometry. Cells were incubated in RPMI-1640 supplemented with 10% fetal bovine serum at 37 °C for 24 hr, and then washed and incubated with 2.5 μg of either Alexa555 labeled siRNA/CH-NPs or siRNA/RGD-CH-NPs in PBS for 20 min at 4 °C. B, Binding of siRNA/RGD-CH-NPs with different concentrations of RGD peptide by flow cytometry. C, Binding of Alexa555 siRNA/RGD-CH-NPs and Alexa555 siRNA/CH-NP in SKOV3ip1 or A2780ip2 cells. Cells were fixed in a chamber slide using 4% paraformaldehyde and then nuclei (blue) were stained with Hoechst 33258 for 10 min, and binding was analyzed by fluorescence microscopy (magnification ×200). Quantitative differences were analyzed by fluorescence intensity of Alexa555 (red)/Hoechst 33358 (blue). Error bars represent s.e.m. *p <0.01. D, Binding of RGD-CH-NPs in SKOV3ip1 or A2780ip2 cells by TEM (N: nucleus, arrows: NPs, bar: 2 μm).

Fig. 3

In vivo delivery of Alexa555 siRNA/RGD-CH-NP. Tumor and organ tissues were harvested after single injection of either control siRNA/CH-NP, Alexa555 siRNA/CH-NP, or Alexa555 siRNA/RGD-CH-NP into SKOV3ip1-bearing mice. Uptake efficiency was determined by the percentage of Alexa555 siRNA-labeled NPs in 5 random fields (original magnification ×200): A, Tumor and C, Various organs. B, Co-localization of Alexa 555 siRNA/RGD-CH-NP or CH-NP (red) and ανβ3 integrin (green) in tumor tissues (original magnification ×200). All of these analyses were recorded in 5 random fields for each slide. Error bars represent s.e.m. *p <0.05.

Fig. 4

Effect of POSTN down regulation following intravenous injection of POSTN siRNA/RGD-CH-NP into SKOV3ip1-bearing mice. A, Western blot analysis was done for POSTN expression in tumor tissue (20 μg of protein used). Quantitative differences were determined by densitometry analysis. B, POSTN expression in tumor tissues was assessed by immunohistochemistry at 24 hr. All of these analyses were recorded in 5 random fields for each slide and quantitative difference was determined by positive/negative expression of cells for staining (magnification ×100). Error bars represent s.e.m. *p <0.05.

Fig. 5

Effect of siRNA/RGD-CH-NP on ovarian cancer growth. Treatment was started 1 week after intraperitoneal injection of tumor cells into mice: A, SKOV3ip1 and B, A2780. Either siRNA-incorporated into CH-NP or RGD-CH-NP was given twice weekly at a dose of 150 μg/kg body weight through intravenous injection. Docetaxel was diluted in PBS and injected intraperitoneally once per week, at a dose of 100 μg, in 200 μl of PBS. Treatment was continued until mice in any group became moribund (typically 4 to 5 weeks depending on tumor cell). The fold change in levels of POSTN mRNA represents the mean of triplicate experiments by qRT-PCR. C, Immunohistochemistry for cell proliferation (Ki67, magnification ×100), microvessel density (CD31, magnification ×100), and TUNEL (bar: 50 μm) was performed on SKOV3ip1-tumor tissues following treatment with POSTN siRNA/RGD-CH-NP or CH-NP. All of these analyses were recorded in 5 random fields for each slide. D, HeyA8 tumor model treated with FAK siRNA/RGD-CH-NP or CN-NP. The fold change in FAK mRNA levels represents the mean of triplicate experiments by qRT-PCR. Error bars represent s.e.m. *p <0.05.

Fig. 6

Therapeutic efficacy of siRNA/RGD-CH-NP against A2780-bearing mice. A, Anti-tumor effect of mouse PLXDC1 siRNA/RGD-CH-NP or CH-NP in the A2780 tumor model. Error bars represent s.e.m. *p <0.05. B, Effect of PLXDC1 siRNA/RGD-CH-NP treatment on apoptosis in the tumor vasculature. Tumor sections were stained with CD31 (red) and TUNEL (green) using double immunofluorescence staining. Colocalization of endothelial cells undergoing apoptosis appears yellow (magnification ×400). All of these analyses were recorded in 5 random fields for each slide and quantitative differences were analyzed by positive expression of TUNEL/CD31. C, Tumor sections were stained with CD31 (red) and PLXDC1 (green) using immunofluorescence staining to examine PLXDC1 silencing in the tumor vasculature (magnification ×400).